Genetic and Phenotypic Diversity and Relations …...2.1. Genetic diversity and relations among...

Chapter 7

Genetic and Phenotypic Diversity and RelationsBetween Grapevine Varieties: Slovenian Germplasm

Denis Rusjan

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51773

1. Introduction

Slovenia is a small central European country situated between the Alps and the Adriatic Sea,at the crossing between Italy (to the West), Austria (to the North), Hungary (to the East) andCroatia (to the South), a historical place of significant grape and wine trading. Grapevineshave been cultivated in Slovenia since ancient times, although the first literal proofs ongrape growing and winemaking date back merely to the Austro-Hungarian Empire [1,2].However, the oldest Vitis vinifera ssp. L. (grape) pips (seeds) found during the archaeologi‐cal excavation of the late Neolithic (Copper Age) pile-dwelling settlement of Hočevarica atthe Ljubljansko barje moor and date back to the 37th/36th century B.C. [3].

The geographical position of the country as well as the regarding climate conditions, and thesocio-political development throughout the nation’s history, have contributed to a diverseassortment of grapevine [1,2]. At least around 100 old and less known varieties are enumer‐ated especially in the western part of Slovenia (Sub Mediterranean) and accompanied by thewidespread European, allochthon, autochthon/landrace and local varieties, a large numberof grapevines is cultivated in Slovenia nowadays [4,5].

The intense trade in grapes and wine, especially due to the Venetians (Venetian Republicfrom the end of 7th to the end of 18th Century; Figure 1), and the varying climatic and geolog‐ical conditions contributed to a great diversity of Vitis vinifera L. varieties. Additionally, themulticulturalism and multilingualism of the area as well as the turbulent historical eventshave contributed to identical genotypes having different names [1,2].

© 2013 Rusjan; licensee InTech. This is an open access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,distribution, and reproduction in any medium, provided the original work is properly cited.

Figure 1. Territories of the Republic of Venice (697–1797). (http://en.wikipedia.org/wiki/File:Repubblica_di_Vene‐zia.png)

Nowadays, Slovenian winegrowers (27.802) produce on 15.973 ha an annual of around 54.3mio litres of wine; 62% whites and 38% reds. Fifty grapevine varieties (Vitis vinifera L.),international, allochthon, local and autochthon as well, are registered in the official Sloven‐ian varietal list, but numerous accessions are still observed and non-descripted in manyparts of Slovenia [1,6]. Although some of them form the basis of the renown regional vari‐eties (‘Rebula’ in Goriška brda, ‘Zelen’ and ‘Pinela’ in Vipavska dolina, ‘Šipon’ in Ljutomer-Ormož etc.), many face extinction, since in some areas only a few plants are reported.However, Slovenia is facing the threat of the rapid erosion of the native/local germplasmon account of the introduction of foreign varieties, such as ‘Merlot’, ‘Cabernet’, ‘Chardon‐nay’ and ‘Pinot’. Very often, however, the problems with the identification of these local,unknown varieties, occur, because of the presence of various synonyms of the same varie‐ty throughout the country [2,6,7].The gene pool of the varieties cultivated in Slovenia iscomposed of old allochthon, autochthonous, domestic/local varieties, which can all be addi‐tionally divided into two groups. The first group consists of commercially used varietieswhich are planted on more than 100 ha each, such as ‘Rebula’, ‘Žametovka’, ‘Zelen’, ‘Pine‐la’, ‘Šipon’, ‘Radgonska Ranina’, and ‘Refošk’. The second group of varieties, such as ‘Vitov‐ska grganja’ and ‘Belina’, are cultivated on less than 100 ha each and are known as localvarieties. Moreover, around 50 well-known and rare varieties/accessions exist in Sloveniatoday; the majority of which have not yet been listed in the International List of Vine Varietiesand Their Synonyms of the O.I.V. (International Organisation of Vine and Wine). Eventhough some morphological and agronomical descriptions related to these accessions ex‐ist, we are talking about mostly unexplored plant material and some of these varieties hadsurvived only in less productive vineyards or owing to germplasm collections. The system‐atically collection of these endangered accessions started in 1980. Nowadays, there are fivegrapevine collections in the whole country: Slap at Vipava, Ampelografski vrt in Krom‐berk, the grapevine collection at Dobrovo in Goriška brda, Meranovo near Maribor and onenear Ormož that include old varieties, clonal candidates and clones (Table1).

The Mediterranean Genetic Code - Grapevine and Olive148

N Variety Synonym/Original name Clone Code

1 'Barbera' SI-36

2 'Beli pinot' Pinot blanc SI-19; SI-20

3 'Chardonnay' SI-21; SI-39; SI-40

4 'Laški rizling' Welschriesling, Graševina SI-11; SI-12; SI-13; SI-41

5 'Istrska malvazija' Malvasia d’Istria, Malvasia istriana SI-37

6 'Pinela' / SI-28

7 'Ranina' Bouvier Traube, Muscat de Saumur SI-4; SI-5; SI-6; SI-7

8 'Ranfol' Štajerska belina SI-38

9 'Rebula' Ribolla gialla SI-30; SI-31; SI-32; SI-33; SI-34

10 'Refošk' Refosco, Teran, Refosco d’Istria SI-35

11 'Renski rizing' Riesling, Rheinriesling SI-22; SI-23; SI-24

12 'Sauvignon' Sauvignon blanc SI-1; SI-2; SI-3

13 'Šipon' Furmint, Moslavac SI-14; SI-15; SI-16; SI-17; SI-18

14 'Traminec' Traminer SI-8

15 'Zelen' / SI-26

16 'Žametovka' Köllner blauer, Kavčina, Žametna črnina SI-25

Table 1. List of varietal clones and clone candidates selected in Slovenia.

Nowadays, varieties can be characterized by several methods: (i) by means of a morphologi‐cal description of plant parts at different phenological stages; (ii) morphometry based on themeasurements of the parameters of the plant organs and (iii) quantitative or qualitativeanalysis of biochemical compounds. Furthermore, traditional methods of varietal descrip‐tion based on vegetative and reproductive (ampelography) parts of plants, contributedgreatly to the full description of the identities and relationships among V. vinifera L. variet‐ies; what also suggests that ampelographic characterization according to the characters putforward by the O.I.V. is the first step in the examination of grapevine varieties/accessions.Several authors described various analyses of primary and secondary metabolites and meth‐ods based on DNA polymorphism as outstandingly useful methods to complete the mor‐phological identification of grapevine varieties [8,9].

2. Slovenian germplasm

One of the problems in the management of these germplasm collections is the use of syno‐nymic and homonymic designations. The lack of order caused by synonyms and homonymsis caused by inadequate documentation and poor preservation of historical facts related togrape growing and trade. The identification and comparison of plant material by ampelo‐graphic methods often results in misinterpretations [10]. In contrast, DNA-based markersare independent of environmental factors and are therefore more appropriate for varietalidentification [9,11]. In the last decade, more than 60 SSR primers from the genomic libraries

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of Vitis vinifera L. have been developed and used for identification purposes [12,13]. The ge‐netic characterization of grapevine varieties (Vitis vinifera L.) using microsatellite markersand ampelometric methods has been done by many countries and regions already. The inte‐gration of the resulting molecular analyses and detection of synonymous grapevine varietieshas already been performed among countries which share a common grapevine assortment,such as Croatia, North Italy, Austria, Germany, France, Spain, Portugal, Greece, etc.[5,9,14,15]. In the last decade many genetic studies of local varieties have been conducted inSlovenia [4,5,15-18] as well, and have consequently revealed the genetic biodiversity ofgrapevine varieties grown and cultivated in the country (Table 2).

Variety / Accession Synonym Homonym Origin

Bela glera / Prosseco, Briška glera G

Beli teran / Vitovska grganja, Vitouska G

Belina Heunish L

Bianchera Erbaluce, Albaluce, Albalucent, Bianco

Rusti, Erbalus, Erbalucente, Uva Rustia

Al

Borgonja bela Istrska malvazija Al

Borgonja rdeča Burgonja istarska, Gamay Beaujolais,

Borgogna

Cipro Al

Briška glera Glera Bela glera, Glera G

Cipro Muškat ruža Porečki, Muškat ruža

omiški, Moscato rosa, Rosenmuskateller

blauer, Likvor, Rdeča muškateljka

Borgonja rdeča Al

Cividin Cividino bianco Al

Cohovka / A

Danijela / G

Dolga petlja / G

Drenik / G

Duranja / Al

Glera Prosecco, Prosekar / Al

Grganc G

Guštana Auguštana G

Istrijanka

Istrska malvazija Malvazija, Malvasia d’Istria, Istarska

malvasia, Istrijanka, Malvasia Istra,

Borgonja bela

/ L

Kanarjola Canaiolo Al

Klarnica Klarnca, Klarna A

Laščina G

Maločrn Piccola nera, Negra Tenera, Petite Raisin Al


Variety / Accession Synonym Homonym Origin

Medena glera / Glera G

Pagadebiti Curzola, Plavina Al

Pergolin / Au

Pinela / Au

Planinka / G

Plavina Brajdica, Curzola, Pagadebiti Plavac mali Al

Pokalca Rdeča rebula, Pocalza, Ribolla Nera,

Schioccoletto, Schiopetino,

Schioppettino, Scoppiettino

Au

Pokov zelen / G

Poljšakica / Au

Pregarc / L

Prosecco Ghera, Glera, Grappolo Spargolo,

Prosecco Balbi, Prosecco Bianco,

Prosecco Tondo, Proseko, Sciorina or

Serprina

Briška Glera, Števerjena Al

Racuk / G

Ranfol Štajerska belina, Štajerka, Urbanka,

Vrbanka, Sremska lipovina, Svetla

belina, Heunisch

Al

Rebula Garganja, Ribolla gialla L

Rečigla / G

Refošk Teran, Refosco, Teranovka, Terrano,

Refosco peduncolo rosso

L

Rožica Rožca G

Sladkočrn / Au

Števerjana / G

Teran Terrano, Refošk / G, L

Teran Istra Terrano, Refošk G, L

Trevolina Al

Vitovska grganja Vitovska, Grganja, Beli Teran Vitouska, Garganja, Beli Refošk,

Racuk

Au

Volovnik Volovna Al, G

Vrtovka / G

Zelen / Au

Zelenika / G

Zunek Cunek G

Table 2. Names, designations and classifications of the less known cultivated grapevine varieties/accessions (Vitisvinifera L.) in Slovenia. Legend: Al – allochthon; Au – autochthon (landrace); L – local (domestic); G – germplasm(grown only in certain gene banks).


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2.1. Genetic diversity and relations among varieties

SSR markers offer some advantages over other molecular markers, including their co-domi‐nant inheritance, hypervariability, and high cross-species transferability [8,13]. Consequent‐ly a large number of markers has been developed for characterisation of grapevine by manyresearch groups [8,12,19] – these markers have provided a very useful and convenient toolfor analysing genetic diversity of grapevine. In order to efficiently manage these conservedlocal germplasm resources and to understand the genetic relationships among them, it isnecessary to characterize the genetic diversity existing in the Slovenian collection and pro‐duction vineyards. However, a majority of cultivated and grown grapevine accessions in theSub-Mediterranean part of Slovenia and a subset of 6 widespread European varieties takenas reference have been genotyped with microsatellite loci in order to: (1) identify and/or dif‐ferentiate varieties, especially those of similar morphological characteristics; (2) assess genet‐ic diversity and relationships among them; (3) and compare these varieties to theirsynonyms [5,15-18] (Figures 2 and 3).

Figure 2. Dendrograms of genotyped grapevine accessions grown in Slovenia [5].


Figure 3. Dendrograms of genotyped grapevine accessions grown in Slovenia [17].

SSR profiles of groups of varieties with similar names but some different morphologicalcharacteristics were compared in order to assess their relationships and resolve existingdoubts on their identity. In order to illustrate the population structure among Slovenian va‐rieties/accessions dendrograms were constructed, which classify the varieties according tothe proportion of shared alleles (Figures 2 and 3). The average similarity of all varieties is34% of shared alleles (Figure 2, left), which is close to the average similarity observed formid-European and Portuguese accessions [5]. Overall, two distinct clusters were obtained,with many sub-clusters. The accessions from the first cluster are related to ‘Laški rizling’(‘Welschriesling’), ‘Rdeča žlahtnina’ (‘Roter Gutedel’, ‘Chasselas red’) from the West Euro‐pean gene pool (Proles occidentalis), but also ‘Rebula’ (‘Ribolla’) known as Proles pontica. An‐other two varieties, ‘Guštana’ and ‘Danijela’, grouped with the first cluster, are both


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characterised by an early grape ripening. The name ‘Guštana’ may describe the ripeningtime of this variety, which is in August. The variety ‘Danijela’ is planted only in gene bankvineyards and its provenance is still uncertain. The variety ‘Picolit’ is cultivated in the west‐ern part of Slovenia (Northern Primorska) and in the north-east part of Italy – in Sloveniatwo distinct, morphologically different types of this variety/accession, ‘Picolit Italia’ and ‘Pi‐colit Vienna’, are known. Microsatellite markers revealed no differences at 21 loci; matchingalso the same allelic profiles at all 7 SSR loci with a variety ‘Picolit’ from Italy [25]. In thesecond cluster, which includes 25 accessions, three groups of synonyms were discovered [5]:‘Vitovska grganja’ = ‘Racuk’, ‘Poljšakica Drnovk’ = ‘Istrska Malvazija’ and ‘Prosecco’= ‘Briš‐ka Glera’ = ‘Števerjana’. ‘Vitovska grganja’ is an old grape variety cultivated in Slovenia inthe winegrowing districts of the Vipavska dolina and Kras and also in the north-east part ofItaly, where it played an important role in the past [1]. The synonymy found between ‘Vi‐tovska grganja’ and ‘Racuk’ could not yet be confirmed despite obtaining identical allelicpatterns, because an accurate morphological characterization of ‘Racuk’ is still lacking. Theallelic profiles of our ‘Vitovska grganja’ have been further compared with the Italian variety‘Vitouska’, recently published by [26] - dissimilarity was revealed at 14 out of 16 loci, andindicating that there is a compatible parent/progeny. The varieties ‘Prosecco’, ‘Briška Glera’and ‘Števerjana’ form another group of synonyms. ‘Prosecco’ and ‘Glera’ have already beenproved to be synonyms on the basis of morphological descriptors and isoenzyme analyses,while ‘Števerjana’ has not previously been considered to be a synonym. The variety ‘Prosec‐co’ takes its name from the village of Prosecco, in the Province of Trieste, where this varietyis also known as ‘Glera’ [26]. The name for the variety ‘Števerjana’ may originate in Štever‐jan, a small village in North-East Italy (Collio), near the Slovenian border. The comparisonof 16 SSR loci of our ‘Prosecco’ with Italian ‘Prosecco tondo’, which was recently analysed[26], revealed no differences - on the basis of this comparison ‘Prosecco’ = ‘Prosecco tondo’ =‘Glera’ but according to [26] ‘Glera’ is related mainly to ‘Prosecco lungo’ and less frequentlyto ‘Prosecco tondo’ [5].

Comparison between the two accessions denominated ‘Glera’ (‘Briška Glera’ and ‘BelaGlera’) included in our analysis [5] revealed differences at 16 out of 21 loci, so they are con‐sidered homonyms. Two varieties, ‘Poljšakica Drnovk’ and ‘Poljšakica Lože’, which were ex‐pected to have the same genetic profile, were different at various analysed microsatelliteloci. The synonymy of ‘Heunisch’ = ‘Ranfol’ = ‘Belina’, which was first mentioned by [2],was also analysed - discrepancies were discovered at 13 loci, but the two varieties share onecommon allele at all 13 loci [5]. Furthermore, the accession ‘Belina Pleterje’ was compared tothe synonymic variety ‘Ranfol Bijeli’ from Croatia using microsatellite data [14] - 8 com‐pared SSR loci data showed the same allelic profiles for all, except VVMD7, where a triallelicprofile was observed. A mutation in the microsatellite sequence of locus VVMD7 was oftenresponsible for identifying grapevine synonyms or related types [19].The SSR profiles of ac‐cessions linked to the designation ‘Zelen’ (‘Zelen Pokov’, ‘Zelen 66’ and ‘Zelen 2.4’) werecompared as well. The differences at several microsatellite loci showed that ‘Zelen’ acces‐sions are a heterogeneous group consisting of several genotypes. The differences were re‐vealed also at the comparison of ‘Zelen’ to ‘Verduzzo’ and ‘Verdicchio’andcan therefore notbe considered/used synonyms.In pairwise comparison, excluding Vitis rupestris L., the great‐


est distances detected (86%) were between accesions ‘Rebula’ – ‘Volovnik’, ‘Dolga petlja’ –‘Guštana’ and ‘Klarnica’ – ‘Pikolit’ (Figure 2).

Subsequent, more detailed study was done in 2011 [17], which included all accessionsfounded in the Sub-Mediterranean part of Slovenia. The observations are reported as fol‐lows [17] (Figure 3): The pairs/groups of vines denominated ‘Duranja’ and ‘Duranja-Pre‐gara’, furthermore ‘Belina’ and ‘Belina1’, ‘Zelen Rup2’, Zelen66’ and ‘Zelen3’ and ‘Malvasia’,‘Istrska Malvasia’ and ‘Malvasia Istriana’ revealed identical genotypes in all SSR microsatel‐lites analysed therefore they are regarded as synonyms. The designations related to ‘Malva‐sia’ very often comprise several types of grapevine varieties which are also morphologicalyheterogenous [20]. Very closely related to ‘Malvasia Istra’ was ‘Borgonja bela’, sharing 19out of 20 alleles, what was also expected according to known ampelographic characteristics.Unexpectedly, vine denominated ‘Laščina 1’ also showed identical SSR profiles as ‘MalvasiaIstra’ but completely different than accession ‘Laščina 2’, what is considered as a designationerror or homonym. In the group of variety ‘Plavina’, the vines signed as ‘Plavina maločrn’and ‘Maločrn’ showed same genotyping results and they are closely related to vine denomi‐nated ‘Plavina Pregara’, what cannot be affirmed for accession signed as ‘Plavina-Pčavina’.‘Plavina-Pregara’ in contrast to ‘Plavina maločrn’ and ‘Maločrn’ resulted in homozygousstate (or heterozygous with a null allele) at locus VVMD7, but ‘Plavina-Pčavina’ was differ‐ent from them in 8 loci out of 10 [17]. The difference in the stage of homozygosity/hetero‐zygosity at locus VVMD7 was also obtained between accessions ‘Glera’ and ‘Briška Glera’and these kind of mutations at locus VVMD7 have previously often been reported [5,9,21].These results of close relatedness suggested that local varieties can be phenotypically partlydifferent types or under-types and that one could have originated from the other throughsomatic mutations. Low relatedness (20% of similarity) was also found between Slovene‘Plavina’ and Croatian ‘Plavina’ genotyped by [20]. ‘Plavina’ was reported the most com‐mon grape variety along the Dalmatian coast and was known as ‘Pagadebiti’ in ‘Curzola’,however in our study the vine denominated ‘Pagadebiti’ is more similar to variety ‘Rebula’(‘Ribolla’) and ‘Kanarjola’ than to ‘Plavina’; as pairwaise comparison revealed only 6% of ge‐netic similarity. The obtained allelic profiles of ‘Pagadebiti’ are also different from the pro‐files of two ‘Pagadebiti’ accessions reported by [21] - ‘Pagadebiti’ translates as “pay thedebt” and so the name may had been used for several very productive grapevine varieties,resulting in many homonyms.

To the group of synonyms related to the designation ‘Glera’ (‘Glera’, ‘Prosecco’, ‘BriškaGlera’, ‘Števerjana’, ‘Beli teran’) the Croatian variety ‘Teran bijeli’ genotyped by [14] wascompared. The comparison of data of 7 SSR loci resulted in identical SSR profile confirmingtheir synonymy. A high genetic difference was revealed between the accessions denominat‐ed as ‘Glera’/’Briška glera’ and ‘Bela glera’ and could be explained with the fact that theterm “Glera” was quite frequently used for white grapevine varieties in the Sub-Mediterra‐nean part of Slovenia in the past [1,24], therefore the obtained homonymy is not a surprise.By comparing allele sizes of 7 SSR loci of the Croatian ‘Muškat ruža Porečki’ [14] with theSlovene ‘Cipro’, identical genotypes were found and therefore synonymy between these two


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varieties was confirmed. On the other hand, ‘Beli muškat’, which is clustered together with‘Cipro’ in the dendrogram, is related to them with a similarity of 60%.

Unexpected the varieties ‘Rožca’ and ‘Zelenika’ showed the same genotype, what can beexplained by the dissemination of scions of the same varieties throughout the winegrowingregions during the past, where the influence of dialects has also put its mark on the denomi‐nation. The variety ‘Rožca’ is found quite close to another white accession signed as ‘Rebulastara’; they share 77% of alleles. Vines denominated ‘Rebula stara’ and ‘Rebula-100’ wereexpected to be closely related but share only 55% of alleles; in common at least one allele ateach locus, which could imply their parent/offspring relation.A genotype of ‘Rebula stara’(=‘Stara Rebula’=‘Rebula old’) perfectly matched at 7 loci with variety ‘Gouais blanc’ (‘Heu‐nisch’). Another interesting finding was the similarity between the vines ‘Grganc’ and ‘Rebu‐la-100’, which showed similar genotypes with exception at locus VVMD5 at which ‘Rebula-100’revealed a triallelic pattern [17].

2.1.1. Grapevine accessions designated ‘Rebula’ and ‘Vitovska’

2.1.1.1. Accessions related to designations ‘Rebula’ and ‘Ribolla’

‘Rebula’ (Vitis vinifera L.) has been one of the most important white grapevine varieties fromthe ancient times down to the present days and has been mostly cultivated in the area ofGoriška brda and Collio where it is still gaining in importance. The first mentions of thename ‘Rebula’ date back to 1299, later 1376,to a deed of sale »Notariorum Joppi« in the areaof the Slovenian Collio [27], whereas the first ampelographic descriptions of the variety arefound in Vinoreja [1] and in Ampelographie [2]. Six different types of the variety ‘Rebula’are enumerated and described (“green rebula”, “yellow rebula”, “less fertile rebula”, “unfer‐tile rebula”, “crazy rebula” and “rebula with smaller and dissected leaf”) [1] what confirmsits biodiversity which decreases with narrow clonal selection. For this variety many syno‐nyms are used - ‘Garganja’, ‘Glera’, ‘Ribolla gialla’, ‘Rebolla’, ‘Ribolla’, ‘Ribolla Bianca’, ‘Ri‐buèle’, ‘Rabuèle’, ‘Rosazzo’, ‘Ribollat’, ‘Raibola’, ‘Ràbola’, ‘Ribuole’ and ‘Gargania’ [6,7,28].DNA analyses regarding accessions of ‘Rebula’ have been already reported [15]. The variety'Rebula' showed a very low similarity (16 %) with other analysed varieties, also with vinesdesigned 'Rebula-100 years' and 'Rebula-old'. The accessions named ‘Prosecco', 'Števerjana','Beli teran' and 'Briška Glera', however, revealed identical genotypes in all 11 SSR microsa‐tellites analysed, and are therefore regarded as synonyms. The varieties 'Rebula' and 'Ribollagialla' revealed an identical SSR profile at 8 out of 9 SSR loci (Figure 4).

Moreover, the genetic identity and relationship among ‘Ribolla gialla’, ‘Rebula’ and ‘Robola’that have been traditionally cultivated in other European Countries (Goriška brda in Sloven‐ia and Kefalonia Island in Greece) grown in private vineyards and in grape collections wereadditionally studied (Table 3)[29]. For this purpose, 35 SSR loci were analysed to fingerprint19 accessions uniformly grown in the three cultivation areas (Friulian Collio, Goriška brdaand Ionian Islands). ‘Ribolla Gialla’ and ‘Rebula’ accessions revealed identical genotype inall 35 analysed SSR markers, therefore are regarded as synonymous. Data proved the exis‐tence of full-sibling relationships between ‘Ribolla gialla’ and ‘Robola’ (Figure 5) [29].


Accession Origin

‘Bela Glera 5/6’

Ampelographical collection - University of Ljubljana – Slovenia

‘Bela Glera 8/1’

‘Glera 1’

‘Glera 2’

‘Glera Medena’

‘Rebula 1’(clone B5) Collection NEBLO – Slovenia

‘Rebula 2’ Ampelographical collection – University of Ljubljana – Slovenia

‘Ribolla 1’Breeding ground GENRRJEVO of RADIKON STANISLAO – Italy

‘Ribolla 2’

‘Ribolla 3’ Breeding ground AZ of RADIKON STANISLAO – Italy

‘Ribolla 4’ Breeding ground CENO of RADIKON STANISLAO – Italy

‘Ribolla 5’ Breeding ground of RADIKON STANISLAO – Italy

‘Robola 1’ Ampelographical collection - University of Tessaloniki – Greece

‘Robola 2’

Kefalonia – Greece

‘Robola 3’

‘Robola 4’

‘Robola 5’

‘Robola 6’

‘Robola 7’

Table 3. List of analyzed accessions and origin area of plant materials related to the designations ‘Rebula’, ‘Ribolla’[29].

At least 9 distinct genotypes resulted from the 19 analysed accessions [29], due to severalcases of detected synonyms (Figure 5) – for example the same genotype was founded amongvines designated ‘Robola 1 and ‘Robola 2’, than between ‘Robola 6’ and ‘Robola 7’, more‐over among five Italian vines designated ‘Ribolla’ accessions (1-5) and three Slovenian acces‐sions (‘Glera 2’, ‘Rebula 1’ and ‘Rebula 2’), ‘Bela Glera 5/6’ with ‘Glera 1’. Besides, severalcases of homonyms revealed, two Slovenian accessions ‘Bela Glera 5/6’ and ‘Bela Glera 8/1’showed different allelic profiles, sharing 48 % of alleles, so did the accessions ‘Glera 1’ and‘Glera 2’(34% of shared alleles). Five cases of homonyms of the group of Greek accessionswere revealed [29]: ‘Robola 1’, ‘Robola 2’ and ‘Robola 6’, ‘Robola 7’ accessions showed allelicprofiles different from other ‘Robola’ accessions. Vine designated ‘Robola 1’ and ‘Robola 2’shared 81 % of alleles with ‘Ribolla Gialla’, ‘Rebula’ and ‘Glera 2’ accessions. In addition[29]report the SSR profiles of five accessions resulted to be unique: ‘Bela Glera 8/1’, ‘Glera Mede‐na’, ‘Robola 3’, ‘Robola 4’ and ‘Robola 5’. After the accurate consideration of the obtainedresults, the studied accessions are clustered into three major groups, but ‘Glera Medena’ and‘Robola 3’ are presented as isolated samples. The cluster 2 presents a major number of ana‐lysed vines, what reveals high homogeneity of the vines cultivated in Italy, as well as in thecomparison with vines cultivated in Slovenia under the name ‘Rebula’ and those fromGreece named ‘Robola’. All these designations from cluster 2 can be perceived as synonyms;


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therefore the original name of this variety is still uncertain. It is demonstrated, that thegroups of Italian and Slovenian vine from cluster 2 share 90% of alleles, suggesting the exis‐tence of a common ancestor[29]. In the case of the other Greek accessions the situation seemsto be more complicated and variability is higher – the largest number of accessions is group‐ed in cluster 1 (‘Robola 4’, ‘Robola 5’, ‘Robola 6’/’Robola 7’ accessions) as they share about75% of alleles, what suggests very close relationships. Grouped in cluster 3, we find the ac‐cessions’ Bela Glera 8/1’, ‘Glera 1’ and ‘Bela Glera 5/6’, which show a similarity of 68% incomparison with cluster 2 (Figure 5). No case of parent-offspring (PO) relationships was re‐vealed among analysed accessions. True-to-type ‘Rebula’ and ‘Ribolla Gialla’ (cluster 2)showed possible full-sibling (FS) relationships with ‘Robola 1’/’Robola 2’ accessions (samecluster), but shared only 55/70 alleles. Among the Greek accessions, ‘Robola 4’, ‘Robola 5’and ‘Robola 6’/’Robola 7’, belonging to a different cluster are related by a FS relationship;‘Robola 4’ and ‘Robola 5’ accessions sharing 68 % of alleles, ‘Robola 4’ and ‘Robola 6’/’Robo‐la 7’ sharing 80 % of alleles and ‘Robola 5’ and ‘Robola 6’/’Robola 7’ sharing 78 % of alleles.Accessions ‘Bela Glera 5/6’, ‘Bela Glera 8/1’ and ‘Glera 1’ shared 48 % of alleles and show aprobable HS relationship [29].

Figure 4. Dendrograms of grapevine accessions related to designations ‘Rebula’ and ‘Ribolla’ using D = 1-(proportionof shared alleles) as coefficient of distance and UPGMA as grouping method [15].


Figure 5. Dendrograms of grapevine accessions related to designations ‘Rebula’ and ‘Ribolla’ using D = 1-(proportionof shared alleles) as coefficient of distance and UPGMA as grouping method [29].

2.1.1.2. Accessions related to designations ‘Vitovska’ and ‘Grganja’

The accessions related to ‘Vitovska’, ‘Vitouska’, ‘Grganja’ and ‘Garganja’ are cultivated indifferent winegrowing areas in Slovenia, Italy and Croatia; therefore their names (syno‐nyms) still cause disorder, because they are often considered indigenous and ancient variet‐ies in the area of cultivation. According to the accurate study [26], which included as muchas 37 nuclear microsatellite locii confirmed the variety ‘Vitouska’ to be the progeny of ‘Mal‐vasia del Chianti’ (syn. ‘Malvasia bianca lunga’, ‘Malvasia lunga’) and ‘Prosecco tondo’. Fol‐lowing this, [30] found an original genotype of ‘Malvasia del Chianti’ with the varieties‘Pavlos’ present in Greece and ‘Maraština’ cultivated in the south coastal region of Croatia(Dalmatia). Already in 1949 it was affirmed that ‘Maraština’ and ‘Malvasia lunga’ are oneand the same variety [31]. In 2012 [18] reported genotyping and phenotyping of accessions(Vitis vinifera L.) cultivated in Slovenia, Croatia and Italia, mostly known as ‘Vito(u/v)ska’and ‘G(a)rgan(i)ja’, while also referred as indigenous or landrace varieties [1,7,28]. The stud‐ied varieties, with an additional focus on variety ‘Vitovska grganja’, were mostly taken fromgermplasm collections: ‘Vitovska grganja’ from Slovenia, ‘Vitouska’, ‘Ribolla gialla’ and‘Prosecco’ from Italy, ‘Garganja’ and ‘Maraština’ from Croatia.


159

Variety / Type / Vine /

Abbreviation

Synonyms and homonyms

(Translation)Cultivation area and notes

‘Vitovska grganja’ 1 (VG) Gerganja1, Vitouska2, Gargania2,

Ribolla gialla2, Grganja3, Garganja3,

Vitovška3, Vitevška3, Vitovka3,

Gorjanska3, Malvazija s piko3, Beli

refošk3, Vrbina3, Vrbovna3, Črna pika3

The vine is approx. 100 years old and

grows in the village Sveto v (address

Sveto 4), Kras (Slovenia) as an individual

pergola-trained vine.

‘Vitovska grganja’ 2 (VG) Gerganja1, Vitouska2, Gargania2,

Ribolla gialla2, Grganja3, Garganja3,

Vitovška3, Vitevška3, Vitovka3,

Gorjanska3, Malvazija s piko3, Beli

refošk3, Vrbina3, Vrbovna3, Črna pika3

The vine is over 110 years old and grows

in the village Briščki (address Briščki 5),

Carso, Italy cultivated as a pergola-

trained individual vine.

‘Vitouska’ (V) Vitovska grganja2, Vitouska2,

Gargania2, Ribolla gialla2

Sampled in the VCR (Rauscedo) selection

of vines from vineyards planted near the

Prosecco village, Italy. The variety is

usually planted and cultivated in Carso

region, north-east part of Italy.

‘Prosecco’ (P) Gljera6, Prosekar6, Prosecco tondo2,

Prosecco bianco2, Gargana2,

Brešanka1,2 (Brescia)

Sampled in the VCR (Rauscedo) clone

VCR101, Italy. The variety is usually

planted and cultivated in Carso region,

north-east part of Italy.

‘Maraština’ (M) Pavlos6, Malvasia del Chianti6,

Malvasia lunga6, Rukatac6, Marinkuša

mala6, Maraškin6, Đerđevina6,

Kukuruz6, Rukac6, Krizol6, Višana6

Sampled in the germplasm collection of

the University of Zagreb, near Split

(Dalmatia, Croatia). The variety is usually

planted and cultivated in Dalmatia,

coastal part of Croatia.

‘Garganja’ (G) Rebula6, Ribolla gialla6 Sampled in the germplasm collection of

the Faculty of agriculture and tourism

Poreč, near Poreč (Croatia). The variety is

usually planted and cultivated in

Croatian part of the Istria peninsula, near

the village Buzet.

‘Ribolla gialla’ (RG) Garganja2, Glera2, Refosco bianco2,

Teran bijeli2, Ribola bijela2, Erbula2,

Jerbula2, Gorička ribola2, Rebolla2,

Ribolla2, Ribolla bianca2, Ribuèle2,

Rabuèle2, Rosazzo2, Ribollat2,

Raibola2, Ràbola2, Ribuole2, Gargania2

Sampled in the VCR (Rauscedo) clone

VCR01, Italy. The variety is planted in

Collio (Italy) presumably also in Goriška

brda (Slovenian Collio), and Istria

(Croatia). Sampled in the collection of

the University of Ljubljana.

Table 4. List of the studied accessions related to the terms ‘Vitovska’, and ‘Garganja’ describing their already usedsynonyms, homonyms and cultivation areas[18]. Indexes: 1-[1]; 2-[28]; 3-[32]; 4-[7]; 5-[30]; 6-[31]


Figure 6. Dendrogram of studied grapevine (Vitis vinifera L.) accessions and of Vitis rupestris based on standard genet‐ic distance [18].

Vines designated as ‘Vitovska grganja 1’ and ‘Vitovska grganja 2’, which have been grownfor more than 100 years in the Slovenian Karst (Kras) revealed identical genotypes in all 11analysed SSR microsatellites. The variety ‘Maraština’, nowadays known as an indigenousgrapevine variety from Central and South Dalmatia (Croatia) surprisingly showed highlyrelated SSR profile as both ‘Vitovska grganja’ cultivated in Slovenia. According to [30,31] thevariety ‘Maraština’ coincides to the varieties ‘Pavlos’ and ‘Malvasia del Chianti’ (Malvasialunga). In our study ‘Maraština’ coincided in seven of eleven SSR loci compared to ‘Malva‐sia del Chianti’ reported by [26], therefore we can conclude only that they are in strong rela‐tionship. Variety ‘Vitouska’ received from VCR Rauscedo (Italy), compared to ‘Maraština’and ‘Vitovska grganja’ 1 and 2, differed in almost all used microsatellites, except at locusVVMD 5 and VVMD36, what additionally confirm the already reported affirmation of null(weak) relationship between both varieties [5,15,26]. Furthermore, considering that [26] af‐firmed the progeny of ‘Vitouska’ with ‘Malvasia del Chianti’ and ‘Prosecco tondo’, a quiteweak match in their locii was found among them in our study.


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2.2. Phenotyping experiences at grapevine varieties cultivated in Slovenia

2.2.1. Phyllometrical tolls for phenotyping

Morphological descriptions of different parts of the plant at different phenological stages,but also morphometry based on the measurements of parameters of plant organs are theoldest methods used in ampelography. Moreover, quantitative and qualitative analyses ofbiochemical compounds were also suggested at varietal description [33]. Despite the techni‐cal advances in biochemical and molecular approaches over the last decades, the descrip‐tions of morphological characteristics remain an important methodology in the descriptionand research of the diversity among species, varieties and clones. The most commune andused methodologies are descriptions according to the OIV codes [34] and ampelometry, es‐pecially phyllometry. The advances of morphological descriptions can be characterised assimple, cheap and applicable in the laboratory or directly in the field. Moreover, the present-day studies focus on finding new parameters in order to differentiate the varieties of vineprecociously, quickly and efficiently [35]. The average vine leaves were reconstructed bymeasuring the proposed morphometrical parameters [36], furthermore a system for directdigitizing of phyllometric parameters from leaves was developed [37]. In grapevine collec‐tions approximately 5-10% of existing varieties are misnamed and even in commercial viti‐culture, misnaming and confusion related to synonyms and homonyms [38] is a frequentphenomena. We started to collect our allochthon, autochthone, local and endangered variet‐ies systematically in 1980 and today we have few germplasm collections around Slovenia.

Additional attention has also been given to varietal phenotyping, especially at those variet‐ies where some misunderstandings in nomination still cause disorders, for example at thevarieties ‘Vitovska grganja’, ‘Refošk’, ‘Rebula’, ‘Glera’, ‘Vitouska’, ‘Garganja’ and others,which are frequently treated as an indigenous, autochthonous or landrace varieties in differ‐ent countries [18]. Some new approaches to accelerate the phenotyping of leaves at differentvarieties were suggested; for example automatic measurements, as well as the possibility oftypical-varietal mature leaf reconstruction [39,40].

2.2.1.1. Phyllometric measurements

Many studies in the last decades demonstrated the importance and contribution of phyllom‐etry in grapevine description and distinction. The method provides around 82 criteria meas‐urements of morphometrical characteristics on an individual leaf. According to a highvariation in these characteristics among leaves of the same variety at least 10 mature, typi‐cally varietal leaves should be picked up, scanned or photocopied, placed in herbarium be‐fore being analysed. In our study we followed that procedure: Herbarium leaves werescanned (with the HP LaserJet M1005scanner) and analysed using AnalySIS image analysisprogram (Soft Imagining System GmbH). The quantitative base variables required the re‐construction of an average typical leaf of different varieties measured following the sugges‐tions from [36] with some modifications. The following morphometrical parameters weremeasured in all leaves sampled per variety: leaf area (LA), length of nerves (L1, L2, L3, L4),distance from the petiole insertion to upper sinuses (OS), distance from the petiole insertion


to lower sinuses (OI), total leaf height (H), total leaf width (W1), distance between the endsof left and right L2 (W2), distance between the basal tooth of the upper lobe (W3), anglesbetween nerves measured from the petiole sinus to the first ramifications of vein (α, β, γ),angles between nerves from petiole sinus to apex of vein (α’, β’, τ), angle at the apex of up‐per lobe (δ1), angle between the apex of L1 and the apexes of L2 (δ2), angle between apex ofL1 and apexes of L5 (δ3), distance between apex and basal tooth of upper lobes (D1), dis‐tance between the ends of L1 and L2 (D2), petiole length (lp), distance from the base of pe‐tiole sinus to the intersection of L3 vein to L4 vein (LO), tooth width at the end of L2 and L4(b1, b2), tooth length at the end of L2 and L4 (h1, h2), distance between the ends of L5 (l),distance between the beginning of L5 (l’) suggested by [41]. Moreover, [42] suggested thefollowing characterizing equations, in which both parts of the same leaf are used separately:Rel.1 = Lp/L, Rel.2 (left side) = L1/L, Rel.3 (right side) = L1/L, Rel.4 (left side) = α+β+γ, Rel.5(right side) = α+β+γ, Rel.6 (left side) = α’+β’+τ, Rel.7 (right side) = α’+β’+τ, Rel.8 (left side) =(OS+OI)/(L1+L2), Rel.9 (right side) = (OS+OI)/(L1+L2), Rel.10 (left side) = OS/L1, Rel.11 (rightside)= OS/L1, Rel.12 (left side) = OI/L2, Rel.13 (right side) = OI/L2 (Figure 7).

Figure 7. The morphometrical characteristics of mature leaves suggested by different authors [36,41,42].

The aim of the study was to investigate the efficiency of ampelographic and morphologicmethods to evaluate the significance of O.I.V. code lists (Paragraph 2.2.2.2) and phyllometryregarding the varietal diversity. In addition, a graphic reconstruction method was appliedand improved to form a typical varietal mature leaf (Figure 9). We evaluated 38 morpholog‐


163

ic and 22 morphometric parameters per leaf. Statistical analysis was carried out, PCA (prin‐cipal component analysis) was performed taking into account equations calculated fromdifferent leaf variables, and the qualitative variables proposed by the O.I.V. were precededto Cluster Analysis. The combination of morphologic descriptors and phyllometric measure‐ments proved to be complementary more than comparable methods. The results of the eval‐uation of O.I.V. descriptors showed a high level of varietal diversity, whereas accession'Barbera type Bovcon' and 'Pokalca' as well as 'Barbera' and 'Syrah' showed some similari‐ties. The most different variety in the group studied according to O.I.V. descriptors was'Touriga national'. The PCA analysis showed the first three components responsible formore than 82% of the discriminating power. The reconstruction determinants were relationsbetween the depth of the lateral sinuses and lateral veins (PC1), relation between first leftlateral vein and central vein, relation between first right lateral vein and central vein (PC2),sum of angles between veins, relation between length of petiole and central vein and depthof the lateral sinuses (PC3). The enumerated relations enable and additionally improve theleaf reconstruction. At the varieties 'Sladkočrn' and 'Refošk' the shallowest lateral sinusesand at 'Tinta pinheira' the deepest ones were observed whereas 'Plovdina' and 'Touriga na‐tional'showed shorter lateral veins (L1d, L1g) in comparison with the main vein (L). Themorphological descriptions and morphometric reconstructions of leaves gave a significantcontribution to understanding the grapevine phenotypical biodiversity.

Figure 8. Dendrogram of grapevine accessions linked to the designations ‘Glera’ and ‘Rebula’ grouped on the basis ofthe 28 morphometric characteristics of mature leaves [40].

Furthermore, [40] studied 28 morphometric characteristics of leaves in detail and their contri‐bution to the similarity among accessions with designations linked to ‘Glera’ and ‘Rebula’.Phyllometric measurements affirmed the genotyping of same varieties studied by [29]. At the


comparison of the groupings in Figure 5 and Figure 8, the measured 28 morphometric char‐

acteristics give suitable results what suggests that phyllometry grants an indispensable, cheap

and credible methodology in the ampelography – varietal description and discrimination.

Figure 9. Graphical reconstruction of leaves of studied grapevine varieties according to determined phyllometric char‐acteristics [39].


165

2.2.1.2. Morphological descriptions

The O.I.V. codes list still remains the most uses and suitable ampelographic tool in contextof grapevine description [34]. There are approx. 147 descriptors; each regarding one signifi‐cant characteristic, which offers a possibility of an accurate description of the main morpho‐logical parts of the vine, for example shoots tip, shoot, leaf, bunch, berry etc. [34]. For basicand relatively fast description of the varieties, the so called the Primary Descriptor list ofOIV codes for Grapevine Varieties and Species Vitis was formed [34], which directs a de‐scription of 14 grapevine characteristics regarding shoots, leaves, bunches and berries. TheOIV descriptors, for some leaf characteristics, overlap with phyllometric parameters: OIV601 = L1, OIV 602 = L2, OIV 603 = L3, OIV 604 = L4, OIV 605 = OS, OIV 606 = OI, OIV 607 = α,OIV 608 = β, OIV 609 = γ, OIV 612 = h1, OIV 613 = b1, OIV 614 = h2, OIV 615 = b2, OIV 618 = l(Figure 7), therefore a collective use of both methodologies upgrades the varietal descrip‐tion. In few studies we used a different number of OIV codes, what depends especially onthe condition of the grapevine, environmental conditions and the scope of the study.

The aim of the first study was the evaluation of selected OIV codes for grapevine descrip‐tion and discrimination – if the selected OIV code descriptorsare enough informative for aprecise varietal discrimination? We sampled from 10 to 12 fully developed leaves from thesixth to ninth node of the shoot per certain variety. For the ampelographic description, shoottips were sampled in the earlier phenological stages and berries were observed during thewhole ripening period. The description and measurements of the particular parts of the vinewere conducted according to the instructions of the individual descriptive codes.

Figure 10. Dendrogram of grapevine varieties grouped according to the morphological evaluation, OIV codes [43].


According to the obtained data (Figure 10) and to the already acquired knowledge of thestudied varieties some expected results revealed – for example similar OIV codes betweenaccessions ‘Refošk’ and ‘Teran Istra’, frequently used as synonyms; or ‘Pokalca’ and ‘Syrah’especially because of shape of the leaves berries. On the other hand the applied OIV codesrevealed some imprecisions as well, for example similarities between varieties ‘Merlot’ and‘Cabernet sauvignon’. Therefore we suggest that the grouping according to the OIV code de‐scriptors should be done and interpreted carefully, as many misunderstandingscould beprovoked. For a comparison and interpretation of the studied accessions we suggest that thereference grapevine varieties should be included always, and as many as possible (also withgreater differences in characteristics).

The further studies was focused on the variations, differences and possible mistakes linkedto the subjective evaluations regarding OIV codes, where different descriptions of the samevariety/accession were collected and compared. Therefore we selected accessions wheremany misunderstanding still provoke - accessions regarding the designations ‘Vitovska’ and‘Garganja’ (Table 5) [18]. The misunderstandings in variety nomination are generally a re‐sult of false and misleading facts, which can partly be explained by neglect in observationsand descriptions of variety characteristics, especially among those with a similar phenotype.

OIV

codeDescriptor VG VGI* V* G* M*,**

001 Young shoot: opening of the shoot tip 5 5 5 5 5

002 Young shoot: distribution of anthocyanin coloration on

prostrate hairs of the shoot tip

2 2**

003 Young shoot: intensity of anthocyanin coloration on

prostrate hairs of the shoot tip

5 6 1 1 5

004 Young shoot: density of prostrate hairs on the shoot tip 7 7 7 3 9, (7**)

005 Young shoot: density of erect hairs on the shoot tip 1

006 Shoot: attitude (before tying) 3 1 1 1 3

007 Shoot: colour of the dorsal side of internodes 2 2 2 2 2

008 Shoot: colour of the ventral side of internodes 1 1 2 2 2

009 Shoot: colour of the dorsal side of nodes 3

010 Shoot: colour of the ventral side of nodes 1

011 Shoot: density of erect hairs on nodes 1

012 Shoot: density of erect hairs on internodes 1

013 Shoot: density of prostrate hairs on nodes 1

014 Shoot: density of prostrate hairs on internodes 1

015-1 Shoot: distribution of anthocyanin coloration of the bud

scale

2 3

015-2 Shoot: intensity of anthocyanin coloration of the bud scale 1 4

016 Shoot: number of consecutive tendrils 1 1 1 1 1


167

OIV


017 Shoot: length of tendrils 5 5

051 Young leaf: colour of upper side of blade (4th leaf) 2,41 3 1 3 2-3, (2-3**)

053 Young leaf: density of prostrate hairs between main veins

on lower side of blade (4th leaf)

7 7 8 7 5-7

054 Young leaf: density of erect hairs between main veins on

lower side of blade (4th leaf)

1

055 Young leaf: density of prostrate hairs on main veins on

lower side of blade (4th leaf)

7

056 Young leaf: density of erect hairs on main veins on lower

side of blade (4th leaf)

1

065 Mature leaf: size of blade 7 7**

067 Mature leaf: shape of blade 3 3 4 4 4, (1,4**)

068 Mature leaf: number of lobes 3 2-3 3 2 5, (3**)

069 Mature leaf: colour of the upper side of blade 7 7**

070 Mature leaf: area of anthocyanin coloration of main veins

on upper side of blade

2 1 2 2 1

071 Mature leaf: area of anthocyanin coloration of main veins

on lower side of blade

2

072 Mature leaf: goffering of blade 5 5 5 5 3, (7**)

073 Mature leaf: undulation of blade between main or lateral

veins

1

074 Mature leaf: profile of blade in cross section 4 5 5 5 1

075 Mature leaf: blistering of upper side of blade 5 3 7 3 3

076 Mature leaf: shape of teeth 3 2,4 4 5 3, (2**)

078 Mature leaf: length of teeth compared with their width 3 5**

079 Mature leaf: degree of opening / overlapping of petiole

sinus

7 5 2 7 7

080 Mature leaf: shape of base of petiole sinus 1 3 1 3 1, (2**)

081-1 Mature leaf: teeth in the petiole sinus 1 1 1 1 1

081-2 Mature leaf: petiole sinus base limited by vein 1 1 1 1 3

082 Mature leaf: degree of opening/overlapping of upper

lateral sinuses

2-3

083-1 Mature leaf: shape of the base of upper lateral sinuses 1

083-2 Mature leaf: teeth in the upper lateral sinus 1 1 1 1 1

084 Mature leaf: density of prostrate hairs between main veins

on lower side of blade

7 1 6 5 7

086 Mature leaf: density of prostrate hairs on main veins on

lower side of blade

7


OIV


087 Mature leaf: density of erected hairs on main veins on lower

side of blade

7 3 2 1 5

093 Mature leaf: length of petiole compared to length of

middle vein

3 5**

094 Mature leaf: depth of upper lateral sinuses 5-7 4 3-5, (5**)

101 Woody shoot: cross section 1-2 2**

102 Woody shoot: structure of surface 1 1-2**

103 Woody shoot: main colour 1,32 1,4**

104 Woody shoot: lenticels 1 1 1

105 Woody shoot: erect hair on nodes 1

106 Woody shoot: erect hair on internodes 1

151 Flower: sexual organs 3 3 3 3, (3**)

152 Inflorescence: insertion of 1st inflorescence 2

153 Inflorescence: number of inflorescence per shoot 1 2**

155 Shoot: fertility of basal buds (buds 1-3) 5 1 5 5, (1**)

202 Bunch: length (peduncle excluded) 5-7 5 7 1 7, (7**)

203 Bunch: width 5-7 5**

204 Bunch: density 7 6 7 5 3, (5-7**)

206 Bunch: length of peduncle of primary bunch 3 4 5 5, (7**)

207 Bunch: lignifications of peduncle 5 5-7**

208 Bunch: shape 2 1 2 1 1-2, (1-2**)

209 Bunch: number of wings of the primary bunch 2 2-3 2 2 3, (2**)

220 Berry: length 3 5 5 3, (3**)

221 Berry: width 3 5 5 3, (3**)

222 Berry: uniformity of size 1

223 Berry: shape 1,2 2 1 2 2, (2**)

225 Berry: colour of skin 1 1 1 1 1, (1**)

226 Berry: uniformity of skin colour 2

227 Berry: bloom 5 7**

228 Berry: thickness of skin 3 7**

229 Berry: hilum 2 2**

231 Berry: intensity of flesh anthocyanin coloration 1 1 1 1 1

232 Berry: juiciness of flesh 2 2**

233 Berry: must yield 7 5**

235 Berry: firmness of flesh 2 1-2 1 2 3

236 Berry: particular flavour 1 5 1 1 1, (1**)

238 Berry: length of pedicel 3 7**

240 Berry: ease of detachment from pedicle 3


169

OIV


241 Berry: formation of seed 3 3 3 3 3, (3**)

242 Berry: length of seeds 5,7 5,7**

243 Berry: weight of seeds 3 3-5**

244 Berry: transversal ridges on dorsal side of seeds 1 1 1

301 Time of bud burst 7 3 3

302 Time of full bloom 5-7

303 Time of beginning of berry ripening (vérasion) 5 5 5

304 Time of full physiological maturity of the berry 5 7**

305 Time of beginning of wood maturity 7

306 Autumn coloration of leaves 1

351 Vigour of shoot growth 7 8 5, (5-7**)

352 Growth of lateral shoot 5

353 Length of internodes 3-5 3**

354 Diameter of internodes 3 3**

401 Resistance to iron chlorosis 1

402 Resistance to chlorides (salt) 1

403 Resistance to drought 7

452 Leaf: degree of resistance to Plasmopara 5

453 Cluster: degree of resistance to Plasmopara 5

455 Leaf: degree of resistance to Oidium 1,3

456 Cluster: degree of resistance to Oidium 1,3

458 Leaf: degree of resistance to Botrytis 5

459 Cluster: degree of resistance to Botrytis 5

501 Percentage of berry set 5

502 Bunch: single bunch weight (g) 3 3 5, (3**)

503 Berry: single berry weight (g) 1-3 3 1, (1-3**)

504 Yield per m2 (kg) 3-53 5, (3**)

505 Sugar content of must 3 3 5 5, (5**)

506 Total acidity of must 3-54 3 5 5, (5**)

508 Must specific pH 3,55 3 7

601 Mature leaf: length of vein N1 (mm) = L1 *** 5 7 5**


603 Mature leaf: length of vein N3 (mm) = L3 *** 5-7 7 5-7**


607 Mature leaf: angle between N1 and N2 measured at the

first ramification (°) = α***

5 7 7**


first ramification (°) = β***

7 7 5**


OIV



first ramification (°) = γ***

5 7 5**

612 Mature leaf: length of tooth of N2 = h1*** 3 7

613 Mature leaf: width of tooth of N2 = b1*** 5 9

614 Mature leaf: length of tooth of N4 = h2*** 3 3-5

615 Mature leaf: width of tooth of N4 = b2*** 5 7

Table 5. Ampelographic description according to O.I.V. descriptor codes and morphometric characteristics of‘Vitovska grganja’ (VG), ‘Vitovska grganija’ (VGI), ‘Vitouska’ (V), ‘Grganja’ (G) and ‘Maraština’ (M) varieties [18]. *-[44];**-[45]; ***-parameters of phyllometry [18]; 1-copper-reddish between veins; yellow around veins; 2-yellow andreddish; 3-0.8-1 kg m-2; 4-total acidity 5.6 - 8.2 g L-1; 5-pH 3.10 - 3.45.

The study was carried out on grapevine (Vitis vinifera L.) varieties cultivated in Slovenia,Croatia and Italia, mostly known as ‘Vitovska’, ‘Vitouska’, ‘Grganja’ and ‘Garganija’ but alsoreferred to as indigenous or landrace varieties from the mentioned countries [1,7,28]. Thestudied varieties, with an additional focus on the ‘Vitovska grganja’ variety, were mostlytaken from germplasm collections: ‘Vitovska grganja’ from Slovenia, ‘Vitouska’, ‘Ribollagialla’ and ‘Prosecco’ from Italy, ‘Garganja’ and ‘Maraština’ from Croatia.

The comparison of the ampelographic descriptions, morphometric and phyllometric charac‐teristics among the ‘Vitovska grganja’ (VG), ‘Vitovska grganija’ (VGI), ‘Vitouska’ (V),‘Grganja’ (G) and ‘Maraština’ (M) varieties is presented in Table 5. The ampelographic de‐scriptions of the VGI, V and G varieties highly differed from the VG and M varieties [18].Similarities between VG and M varieties were observed in the characteristics of young andmature shoots, the opening shoot tips showed similar anthocyanin coloration and a similarintensity and coloration of prostrate hairs. Similarities between VGI and M varieties have tobe underlined, especially in the size of young and mature leaves, dark green coloured upperside of the blade, both convex shaped sides of the teeth. The latter descriptor was in contrastto the report of [45], where both sides were described as straight. Moreover, [45] describedthe shape of the petiole base sinus of the M variety as ‘U’-shaped whereas in [44] it is shownbrace-shaped, what is also in accordance with our observations on VG variety. Both M andVG varieties were characterized by high density prostrate hairiness between the main veinson the lower side of the blade; however, hair density differed at other blades/vein parts. Ourobservations regarding the depth of the upper lateral sinuses of VG variety coincided withthose mentioned by [45] for the M variety. In our study, M variety had a lower fertility ofbasal buds (1-3) compared to VG variety and also to the results reported by [45].

A comparison of bunch and berry characteristics among the studied varieties were also re‐ported [18], what partly explains the reasons of the misuse of the variety nominations due tothe rather similar phenotypes. All studied varieties have a cylindrical or conical shape of thebunches, obloid and globose berries, and a bunch length ranging between 160 and 200 mmwith the exception of M and VG varieties, where berries are distinctively shorter and nar‐rower. The description of single bunch and berry weight, as well as yield of the M variety


171

reported by [45], coincided with our description for the VG variety. On the other hand,many significant differences were observed among the studied varieties, also between VGand M variety. Compared to M variety, the VG variety blooms later, has thinner berry skin,higher must yield, less firm flesh, shorter length of pedicle, lower sugar and total aciditycontent and lower specific pH level of the must. Moreover VG also has later bud bursting,but reaches full physiological maturity of the berry earlier. [18] found also some similaritiesamong the studied varieties, specifically in morphometrical characteristics of mature leaves.Significant similarities were observed between our measurements of the VG variety and da‐ta cited by [45] for the M variety, which only differed in ‘α’ and ‘β’ angles. The comparisonof phenotypic characteristics of the studied varieties showed many similarities, which sug‐gests that a consistent attention to the ampelographic distinguishing among varieties, espe‐cially among those with the same designations or synonyms, has to be emphasized in thefuture.

3. Conclusions

The grapevine genepool is particularly vulnerable in the marginal areas of its distributionrange. Many grapevine varieties have been already described and their genotypes deter‐mined; but many local grapevine accessions remain unidentified and their ampelographiccharacteristics overlooked. Accurate ampelographic description of variety should be done withcombined methodologies which involve morphological description and DNA analyses. In thelast decade a great effort was given to the DNA analyses – genotyping of the grapevine varieties,and quite soon revealed that DNA analyses is simply not enough to answer all questions aboutvarietal discrimination and description. Parallel to the development of genetic techniques, themorphological and morphometric evaluations have fall into oblivion. Although, microsatel‐lites are very powerful means of identifying synonyms in germplasm collections, they haveto be supported with morphological descriptions, what was also demonstrated in our research‐es. Only such works will allow a characterization of a large number of varieties/accessions andwill contribute to improve the organization of grapevine collections and the possibility ofexchanging true-to-type material. The presented works are a first step towards true-to-typeidentification, which will facilitate the registration of varieties and allow growers to be sureof the value of their products. Starting true-to-type variety identification by comparing re‐sults with neighbouring, historically linked areas, will significantly clarify the confusion innomenclature and help determine the origin and relationships among varieties over the wholearea. In the future all the obtained results from different studies should be upgraded to thesame database for grapevine varieties, to obtain a total overview of World germplasm, whatmay be the key to stop the erosion of the many varieties.


Author details

Denis Rusjan*

Address all correspondence to: [email protected]

Biotechnical Faculty, University of Ljubljana, Slovenia

References

[1] Vertovec, M. (1845). Vinoreja za Slovence. Ajdovščina: Agroind Vipava.

[2] Goethe, H. (1887). Berlin: Handbuch der Ampelographie. Beschreibung und Klassifika‐tion der bis jetzt kultivierten Rebenarten und Trauben-varietäten mit Angabe ihrer Syno‐nyme, Kulturverhältnisse und Verwendungsart. Berlin: P. Parey.

[3] Tolar, T., Jakše, J., & Korošec-Koruza, Z. (2008). The oldest macroremains of Vitisfrom Slovenia. Vegetation history archaeobotany, 17(1), 93-102.

[4] Tomažič, I., & Korošec, Koruza. Z. (2003). Validity of phyllometric parameters usedto differentiate local Vitis vinifera L. cultivars. Genetic resources and crop evolution,50(7), 779-787.

[5] Štajner, N., Korošec-Koruza, Z., Rusjan, D., & Javornik, B. (2008). Microsatellite geno‐typing of old Slovenian grapevine varieties (Vitis vinifera L.) of the Primorje (coastal)winegrowing region. Vitis, 47(4), 201-204.

[6] Plahuta, P., & Korošec-Koruza, Z. (2009). x sto vinskih trt na Slovenskem. Ljubljana:Prešernova družba

[7] Hrček, L., & Korošec-Koruza, Z. (1996). Sorte in podlage vinske trte: ilustrirani prikaztrsnega izbora za Slovenijo. Ptuj: Slovenska akademija Veritas

[8] Thomas, M. R., & Scott, N. S. (1993). Microsatellite repeats in grapevine reveal DNApolymorphisms when analysed as sequence-tagged sites (STSs). Theoretical and Ap‐plied Genetics, 86(8), 985-990.

[9] Crespan, M. (2004). Evidence on the evolution of polymorphism of microsatellitemarkers in varieties of Vitis vinifera L. Theoretical and Applied Genetics, 108(7),231-237.

[10] Dettweiller, E. (1993). Evaluation of breeding characteristics in Vitis. Influence of cli‐mate on morphologic characteristics of grapevines. Vitis, 32, 249-252.

[11] Cipriani, G., Frazza, G., Peterlunger, E., & Testolin, R. (1994). Grapevine fingerprint‐ing using microsatellite repeats. Vitis, 33, 211-215.


173

[12] Bowers, J. E., Dangl, G. S., Vignani, R., & Meredith, C. P. (1996). Isolation and charac‐terization of new polymorphic simple sequence repeat loci in grape (Vitis vinifera L).Genome, 39(4), 628-633.

[13] Sefc, K. M., Lefort, F., Grando, M. S., Scott, K., Steinkellner, H., & Thomas, M. (2001).Microsatellite markers for grapevine: A state of the art. In: Roubelakis-Angelakis KA.(ed.) Molecular Biology & Biotechnology of the Grapevine. Dordrecht: Kluwer Aca‐demic Publishers , 433-466.

[14] Maletić, E., Sefc, M. K., Steinkellner, H., Karoglan, Kontić. J., & Pejić, I. (1999). Geneticcharacterization of Croatian grapevine cultivars and detection of synonymous culti‐vars in neighboring regions. Vitis, 38(2), 79-83.

[15] Rusjan, D., Jug, T., & Štajner, N. (2010). Evaluation of genetic diversity: which of thevarieties can be named ‘Rebula’ (Vitis vinifera L.)? Vitis, 49(4), 189-192.

[16] Kozjak, P., Korošec-Koruza, Z., & Javornik, B. (2003). Characterisation of cv. Refošk(Vitis vinifera L.) by SSR markers. Vitis, 42(2), 83-86.

[17] Štajner, N., Rusjan, D., Korošec-Koruza, Z., & Javornik, B. (2011). Genetic characteri‐zation of old Slovenian grapevine varieties of Vitis vinifera L. by microsatellite geno‐typing. American Journal of Enology and Viticulture, 62(2), 250-255.

[18] Rusjan, D., Pipan, B., Pelengić, R., & Meglič, V. (2012). Genotypic and phenotypic dis‐crimination of grapevine (Vitis vinifera L.) varieties of the ‘Vitovska’ and ‘Garganja’Denominations. European Journal of Horticultural Science, 77(2), 84-94.

[19] Lefort, F., & Roubelakis-Angelakis, K. A. (2001). Genetic comparison of Greek culti‐vars of Vitis vinifera L. by nuclear microsatellite profiling. American Journal of Enologyand Viticulture, 52(2), 101-108.

[20] Lacombe, T., Boursiquot, J. M., Laucou, V., Dechesne, F., Varès, D., & This, P. (2007).Relationships and Genetic Diversity within the Accessions Related to Malvasia Heldin the Domaine de Vassal Grape Germplasm Repository. American Journal of Enologyand Viticulture, 58(1), 124-131.

[21] Riaz, S., Garrison, K. E., Dangl, G. S., Boursiquot, J. M., & Meredith, C.P. (2002). Ge‐netic divergence and chimerism within ancient asexually propagated winegrape cul‐tivars. Journal of the American Society for Horticultural Science, 127(4), 508-514.

[22] Caló, A., Costacurta, A., Maras, V., Meneghetti, S., & Crespan, M. (2008). Molecularcorrelation of Zinfandel (Primitivo) with Austrian, Croatian, and Hungarian culti‐vars and Kratošija, an additional synonym. American Journal of Enology and Viticul‐ture, 59(2), 205-209.

[23] Muganu, M., Dangl, G., Aradhya, M., Frediani, M., Scossa, A., & Stover, E. (2009).Ampelographic and DNA characterization of local grapevine accessions of the Tusciaarea (Latium, Italy). American Journal of Enology and Viticulture, 60(1), 110-115.


[24] Zulini, L., Fabro, E., & Peterlunger, E. (2005). Characterisation of the grapevine culti‐var Picolit by means of morphological descriptors and molecular markers. Vitis,44(1), 35-38.

[25] Crespan, M., Crespan, G., Giannetto, S., Meneghetti, S., & Costacurta, A. (2007). Vi‐touska’ is the progeny of ‘Prosecco tondo’ and ‘Malvasia Bianca lunga’. Vitis, 46(4),192-194.

[26] Crespan, M., Cancellier, S., Chies, R., Giannetto, S., Meneghetti, S., & Costacurta, A.(2009). Molecular contribution to the knowledge of two ancient varietal populations:‘Rabosi’ and ‘Glere’. Acta Horticulturae ISHS, 827(1), 217-220.

[27] Cosmo, I., & Polsinelli, M. (1957). Ribolla gialla. Roma: Annali della sperimentazioneagraria.

[28] Caló, A., Scienza, A., & Costacurta, A. (2006). Vitigni d’Italia. Bolognia: Edagricole-Edizioni Agricole Srl.

[29] De Lorenzis, G., Failla, O., Scienza, A., Rusjan, D., Nikolaou, N., Vouillamoz, J., &Imazio, S. (2012). Study of genetic differences among ‘Ribolla Gialla’, ‘Rebula’ and‘Robola’ (Vitis vinifera L.) respectively from North Eastern Italy, Northern Balkansand Ionian Islands (under review).

[30] Šimon, S., Maletić, E., Karoglan Kontić, J., Crespan, M., Schneider, A., & Pejić, I.(2007). Cv. Maraština a new member of the Malvasia group Mediterranean Malva‐sias: conference proceedings. October 3-6, 2007, Salina, Italija.

[31] Bulić, S. (1949). Dalmatinska ampelografija., Zagreb: Poljoprivredni nakladni zavod.

[32] Blažina, I., Štolfa, D., Korošec-Koruza, Z., & Cerkvenik, D. (1990). Vitovska grganja’(Vitis vinifera L. ‘Vitovska grganja’) in domača ‘Grganja’ (Vitis vinifera L. ‘Domačagrganja’). Zbornik Biotehniške fakultete, 1, 10-14.

[33] Di Stefano, R. (1996). Metodi chimici nella caratterizzazione varietale. Rivista di Viti‐coltura é Enologia, 49(1), 51-56.

[34] OIV. (2008). 2nd Edition of the OIV descriptor list for grape varieties and Vitis spe‐cies. Paris: Organisation Internationale de la Vigne at du Vin.

[35] Schneider, A. (1996). Grape variety identification by means of ampelographic andbiometric descriptors. Rivista di Viticoltura é Enologia, 49(1), 11-16.

[36] Martínez, M. C., & Grenan, S. (1999). A graphic reconstruction method of an averageleaf of vine. Agronomie, 19, 491-507.

[37] Alessandri, S., Vignozzi, N., & Vignini, A. M. (1996). AmpeloCADs (AmpelographicComputer-Aided Digitizing system). An integrated system to digitize, file and proc‐ess biometrical data from Vitis spp. Leaves. American Journal of Enology and Viticul‐ture, 47(3), 257-267.


175

[38] Weihl, T., & Dettweiler, E. (2000). Differentiation and identification of 500 grapevine(Vitis vinifera L.) cultivars using notations and measured leaf parameters. Acta Horti‐culturae, 528, 35-41.

[39] Pelengić, R., & Rusjan, D. (2010). Efficacy of ampelographic and phyllometric toolsfor the validation of grapevine Vitis vinifera L. biodiversity in Slovenia. InternationalJournal Of Food, Agriculture & Environment, 8(3&4), 563-568.

[40] Pelengić, R. (2012). Ampelografski model za identifikacijo sort žlahtne vinske trte(Vitis vinifera L.). PhD thesis. University of Ljubljana; (under review).

[41] Alleweldt, G., & Dettweiler, E. (1989). A model to differentiate grapevine cultivarswith the aid of morphological characteristics. Rivista di Viticoltura é Enologia, 42(1),59-63.

[42] Santiago, J. L., Boso, S., Martínez, M., Pinto-Carnide, O., & Ortiz, J. M. (2005). Ampe‐lographic comparison of grape cultivars in Northwestern Spain and Northern Portu‐gal. American Journal for Enology and Viticulture, 56(3), 287-290.

[43] Pelengić, R. (2006). Ampelographic and ampelometric description of red grape vari‐eties (Vitis vinifera L.) from collection Ampelografski vrt. Graduation thesis.Universityof Ljubljana.

[44] The European Vitis Database. (2011). Institute for Grapevine Breeding: Geilweilerhof.,http://www.eu-vitis.de/index.php, accessed 10 November 2011.

[45] Maletić, E. (1993). Ampelographic investigation of cvs Maraština, Bogdanuša, Vuga‐va and Pošip (Vitis vinifera L.) in conditions of Ravni Kotari, region Coastal Croatia.MSc thesis.University of Ljubljana.


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